The conversion of HIV genomic RNA into DNA comprises multiple steps catalyzed by the viral enzyme, reverse transcriptase (RT). Accordingly, RT is multifunctional, with both DNA polymerase and ribonuclease H (RNase H) activities. Many inhibitors of HIV RT DNA polymerase activity have been discovered including ten drugs in clinical use, whereas few inhibitors of RT RNase H have been identified. Certain N-acyl hydrazones (NAH) are reasonably potent inhibitors of HIV RT RNase H activity. This Program Project component will characterize and optimize the RNase H inhibitory properties of NAH. This project thus comprises three specific aims: (1) To evaluate the mechanisms of NAH inhibition of HIV RNase H. The mechanism of inhibition of target enzyme activity impacts on the development of potent inhibitors of this target. Studies will include steady-state and transient kinetic analyses, the role of metal-binding in inhibition, comparison of NAH inhibition of polymerase-dependent and polymerase-independent RNase H activities, and effect of inhibitors on strand-transfer (novel reagents prepared by Program member R. Jones will be invaluable in these studies). 'Real-time' PCR analysis of intracellular HIV DNA synthesis in the presence of NAH (in collaboration with Program member S.H. Hughes) will assess inhibitory mechanisms in situ. (2) To optimize the antiviral and inhibitory properties of NAH. This will involve the use of traditional and parallel library synthesis to prepare new NAH analogs altered in one or more of the three 'pharmacophore' regions of the molecule to improve inhibitory potency and reduce cytotoxicity. Refinement of NAH structure will involve molecular modeling/docking and quantitative structure-activity analyses, but will primarily be based on exact structural information obtained from X-ray crystallography of RT-NAH complexes (in collaboration with Program director E. Arnold). This structural information will enable rational design of new highly potent analogs. (3) To evaluate the antiviral and resistance properties of NAH inhibitors. Low toxicity inhibitors will be used to generate resistant HIV in vitro. NAH-specific mutations identified will be further characterized by X-ray crystallography of mutant recombinant protein (in collaboration with Arnold and Hughes). The proposed studies will develop novel antiviral agents against HIV RT-associated RNase H, a critical yet under-explored HIV target.